Is blood-brain barrier a probable mediator of non-invasive brain stimulation effects on Alzheimer’s disease?

Alzheimer’s disease (AD) is a complex neurodegenerative disease with no existing treatment leading to full recovery. The blood-brain barrier (BBB) breakdown usually precedes the advent of first symptoms in AD and accompanies the progression of the disease. At the same time deliberate BBB opening may be beneficial for drug delivery in AD. Non-invasive brain stimulation (NIBS) techniques, primarily transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), have shown multiple evidence of being able to alleviate symptoms of AD. Currently, TMS/tDCS mechanisms are mostly investigated in terms of their neuronal effects, while their possible non-neuronal effects, including mitigation of the BBB disruption, are less studied. We argue that studies of TMS/tDCS effects on the BBB in AD are necessary to boost the effectiveness of neuromodulation in AD. Moreover, such studies are important considering the safety issues of TMS/tDCS use in the advanced AD stages when the BBB is usually dramatically deteriorated. Here, we elucidate the evidence of NIBS-induced BBB opening and closing in various models from in vitro to humans, and highlight its importance in AD.

BBB in AD are necessary to better understand their mechanisms and to boost their effectiveness in AD. Moreover, such studies are important considering the safety issues of NIBS use in AD stages when the BBB is damaged. Here, we elucidate the evidence of NIBS-induced BBB changes in humans, animals, and cellular models and highlight its importance in AD.
Blood-brain barrier role in Alzheimer's disease: etiology, pathophysiology, and modeling The BBB is a semi-permeable membrane within mature brain microvessels, protecting neurons from factors presenting in the systemic circulation. The BBB is formed by the components of the neurovascular unit-vascular cells (endothelial cells, pericytes, smooth muscle cells), glia (astrocytes, oligodendroglia, microglia), neurons and extracellular matrix 11,14,15 . Endothelial cells form tight junctions-a physical barrier, whereas other cell types provide signaling among the cells. The BBB breakdown in AD is an increase in vascular permeability associated with a decrease in the expression of tight junction proteins ( Fig. 1; ref. 16 ). The BBB breakdown accompanies even healthy aging, while in patients with mild cognitive impairment and AD dementia, the BBB breakdown is accelerated 17 . The BBB-associated changes may be an early AD biomarker 18,19 . Notably, brain endothelial cells, among all vascular cells, demonstrate their higher vulnerability in AD, based on gene expression data 20 . Although there is no etiotropic treatment, the search for AD biomarkers at early stages is important 21 because the early diagnosis may serve as a motivation to correct a lifestyle and decrease possibly modifiable risks. For example, positron emission tomography (PET) studies have already shown that patients with mild cognitive impairment have reduced glucose uptake across the BBB and, therefore, disintegrated BBB in young subjects may be considered an early marker of AD 22,23 . The BBB leakage is associated with changes in tight junctions, the overall BBB permeability index, transendothelial resistance, etc 24,25 ., and all such alterations were reported in AD pathology ( Fig. 1) 26,27 .
Considering the key role of the BBB in pharmacological brain therapy in general, there are already plenty of approaches allowing to assess the BBB permeability, such as (1) in vitro transwell models, allowing BBB permeability assessment to fluorescent tracers and transendothelial electrical resistance (TEER) 28-31 ; (2) in vivo techniques like microdialysis, used for the analysis of the BBB permeability changes during or after an intervention (e.g., NIBS, drug, injection of a pathogenic ADassociated protein) 32 ; (3) specific type of positron emission tomography: F-2-fluoro-2-deoxy-d-glucose-PET in humans 33,34 .
The BBB disruption and Aβ propagation seem to be intertwined, but they are mostly parallel processes. It is known that in AD, matrix metalloproteinases may digest tight junction and adherent proteins of the BBB 27 , decreasing the levels of tight junction proteins in the brain, and resulting in the physical breakdown of the BBB 26 . Aβ in AD also alters the tight junction proteins content, destroying the BBB 29,35 . Recent preliminary data have demonstrated that repetitive TMS (rTMS) simultaneously diminishes the matrix metalloproteases level and improves cognitive functioning in MCI patients 36,37 . AD-induced BBB abnormalities associated with changes in matrix metalloproteinase (MMP) and tight junction protein content are tightly connected with amyloid, and it makes these parameters sensitive to strong therapeutic stimuli. Brain stimulation is supposed to belong to a number of stimuli that may affect AD progression via the BBB disruption. Better understanding of the pathogenic proteins associated with AD-either peripheral or central-is needed for better understanding of the BBB role in AD. A collaborative work between clinicians and scientists is required to piece together knowledge on AD progression and the BBB breakdown.
Non-invasive brain stimulation as a possible treatment in Alzheimer's disease Among techniques most widely used to target neuronal dysfunction in neurodegeneration, TMS and tDCS are in precise focus of the perspective. NIBS-mediated modulation of cognitive functions in AD is commonly considered to be connected with the changes in neural activity 13,38 , while vascular aspects are mostly overlooked. At the same time, vascular pathology in AD is highly amyloid-dependent considering (1) probable amyloid peripheral origin, (2) its high concentration in the brain blood flow, (3) co-localization of classic and diffuse amyloid plaques with the brain vessels, and (4) known biochemical and functional amyloid effects on the BBB cells 29,[39][40][41] . Thus, NIBS amyloid targeting may be considered in a tight association with probable NIBS effects on the brain vascular system. Among attractive targets for TMS/tDCS application in AD are the frontal lobe, specifically, Broca's area, dorsolateral prefrontal cortex, parietotemporal lobe (Wernicke's area), bilateral parietal somatosensory association cortices, temporal lobe 42,43,44,45 . tDCS is a safe and easy-applicable method, which is widely studied in AD. Different tDCS protocols applied in AD patient cohorts improved recognition memory and general cognitive abilities, but the results were variable among studies [45][46][47] . A randomized, placebo-controlled study in AD patients revealed no effect of temporal cortex tDCS on cognition 48 , while the studies confirming temporal lobe tDCS effects in AD differed in study design, protocols and cognitive tasks 45,49 .
TMS is another widely used NIBS approach with better spatial accuracy compared to tDCS. The use of TMS at the earlier stages of AD was demonstrated as a prospective tool for treatment 38,50 . According to Dong and co-authors' review 42 , TMS modulates cognitive functions based on the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog), but does not affect Mini-Mental State Examination (MMSE) score. The review articulates the importance of TMS frequency in AD treatment: highfrequency rTMS shows big effect, at least based on ADAS-Cog scale. Multiple TMS/tDCS studies both in patients and in healthy participants were followed by their unsuccessful replications [51][52][53][54] . We hypothesize that replication failures related to TMS/tDCS application in AD may be in part connected with our poor understanding of their non-neuronal effects, including effects on the BBB.
Possible influence of transcranial magnetic and electric stimulation on blood-brain barrier in Alzheimer's disease Here, we focus on the BBB as a probable mediator of NIBS effects in AD. The BBB-mediated random NIBS effects may be connected with changes in tight junction protein expression, transendothelial resistance, permeability to molecules, etc. We suggest that in AD, TMS/TES-induced non-neuronal changes may be an important mediator of TMS/TES effects. Apart from the immediate changes of the BBB permeability, there is evidence of the endothelial intracellular parameters' change, including mitochondria abnormalities and FGF-2-related changes 55 . Interestingly, magnetic field effects on endothelial cells may be associated with the same factors [56][57][58] . Electric field effects on endothelial cells are mediated by vascular endothelial growth factor (VEGF) receptor signaling, activation of ATP-receptor P2Y, Ca 2+ and NO concentration transients [59][60][61] . These factors are highly involved in AD pathology [62][63][64] . Importantly, in AD mouse model, BBB endothelial cells produce a variety of factors, such as thrombin, vascular endothelial growth factor (VEGF), angiopoietin-2, tumor necrosis factor (TNF), transforming growth factor, interleukin (IL) IL-1, IL-6, IL-8, monocyte chemoattractant protein-1, hypoxia-inducible factor-1, MMPs, and integrins that may promote AD pathogenesis 65 . A systematic analysis of NIBS effects on AD-associated factors in endothelial cells has not yet been conducted, but some data regarding neurons and glial cells are already available. It was reported, for example, that 10 Hz rTMS may significantly modulate anti-and proinflammatory marker expression either in primary astrocytes exposed to oxygenglucose deprivation/ reoxygenation and make a neuroprotective effect 66 . For instance, the expression of IL-10 gene, associated with anti-inflammation, significantly increased in the astrocyte culture after 10 Hz rTMS 66 . Thus, as interleukin-10 is a supporting factor for the endothelium 67 , we hypothesize that NIBS might induce both direct and indirect effects on the endothelial cells, at least within a neurovascular unit. All this legitimates the concerns about the necessity to test the BBB integrity before/after TMS/TES application in AD. As the BBB state and AD progression are connected 11,68 , we hypothesize that in the earlier stages of AD, TMS/TES is applied to a brain with a less damaged BBB. In contrast, at later stages, TMS/TES influences already a not-integrative BBB (Fig. 1). Thus, for the early AD stages, the possibility of short-term NIBS-induced BBB opening may be helpful for drug delivery, while for the later AD stages, Fig. 1 Blood-brain barrier in health and in Alzheimer's disease. a The blood-brain barrier is composed of endothelial cells, forming tight junctions, pericytes, astrocytes, and neurons. b The blood-brain barrier (BBB) state in a healthy person (left) and in Alzheimer's disease (AD) patient (right) is shown. At the top of the image, AD-associated BBB changes are shown, such as microvascular reduction, degradation of endothelial cells, loss of pericyte number, capillary coverage and capillary basement membrane rearrangement, etc. BBB damage activates the processes of neuroinflammation reflected by the peripheral macrophage and neutrophil infiltration. At molecular level (in the figure, below), protein homeostasis in health (left) and in AD (right) is given: decrease in aquaporin 4 (AQP4), low-density lipoprotein receptor-related protein 1 (LRP1), glucose transporter 1 (GLUT1), P-glycoprotein (P-gp) and increase in receptor for advanced glycation end products (RAGE) levels. For details, see refs. 38,80,84 . BBB restoration can be considered a goal, and an excessive BBB opening is a safety issue. Here, we discuss the experimental works dedicated to the effects of magnetic (Table 1) and electric  (Table 2) stimulation on endothelial cell cultures and the BBB cells, in vivo animal models, and in humans and argue for their relevance for TMS/TES neuromodulation in AD.
NIBS effects on blood-brain barrier opening. NIBS-induced BBB opening may be promising for drug delivery in AD, and it has already been tested for the hippocampus 69,70 and prefrontal cortex 71 using FUS. At the same time, BBB opening may be a safety concern in AD. Thus, one should test whether TMS/tDCSinduced BBB opening is not associated with pathological features of the BBB deterioration characteristic for AD (e.g. pericyte, endothelial, and neuronal degeneration; the BBB transporter function abnormalities; inflammation; accumulation of toxic agents) 11 . The TMS/tDCS effects on BBB opening are summarized below. The effect of magnetic stimulation on the BBBassociated mechanisms was investigated from the very beginning of TMS use. In the early 1990 study, Ravnborg and co-authors showed that single-pulse TMS in a rat did not change the BBB permeability 72 . However, since that time, there were several successful efforts to open the BBB using TMS 73,74 . TMS increased BBB permeability both in animals (in rats, at a low frequency but not at a high frequency) and in patients with malignant brain tumors 73 . Recently, in an attempt to trigger the BBB opening in a rat model, repetitive low-frequency TMS was applied to the rat's right hemisphere, and fluorescent angiography revealed ̴ 18% increased permeability 15 min after the stimulation onset. After the other 15 min, vascularization gets normalized 74 . It is worth noting that to be effective, NIBS-induced BBB opening interventions should be developed considering the pharmacokinetics, as the peak concentration of a drug in the brain may be achieved only in 2-3 h after the drug administration 75 . Also, there is significant evidence to support that tDCS may also modulate BBB integrity (see the review 76 ). It was reported that tDCS temporarily increased the BBB permeability in a rat brain, and this effect was mediated by NO 61 . Nitric oxide synthase (NOS) inhibitor ameliorated tDCS-induced BBB permeability 61 . To our knowledge, there is the only human study, investigating BBB changes after 1 milliampere (mA) tDCS in healthy participants, which did not reveal any BBB changes using diffusion-weighted MRI 77 .
NIBS effects on blood-brain barrier closure. Interestingly, there are also data supporting the BBB closing effect of NIBS. For instance, in the recent rTMS work on a rabbit eye model, it was shown that the expression of a tight junction protein ZO-1 increased after repetitive magnetic stimulation 78 . The main limitation of this study is that stimulation was applied to the cornea of a rabbit keratopathy model and, thus, targeted corneal epithelial barrier functions, not the BBB. However, the corneal epithelial and endothelial tissues in the brain vessels are similar in terms of ZO-1 expression, which may indirectly indicate that TMS might also strengthen the BBB in certain circumstances. Another study using electroconvulsive therapy on the Gunn rat model showed an increase in tight junction protein claudin-5 expression and astrocytic coverage of the brain blood vessels after stimulation 79 . The limitation of this study is that one may not directly compare electrical, magnetic, and electroconvulsive therapy due to the different principles behind them. In the other recent work on the photothrombotic stroke rat model, the BBB damage was successfully mitigated by theta-burst TMS: BBBassociated tight junction protein expression, morphology, and perfusion of vessels preserved 80 . However, the direct comparison of the BBB modulation mechanisms in stroke and in AD is challenging, as the BBB disruption may be triggered by different factors 81 . To our knowledge, there are no studies directly reporting that NIBS in AD will lead to the BBB closure or may hamper the transport of pharmacotherapeutic agents to the brain.
There are also a few studies trying to mitigate the BBB disruption using TMS/TES where no clear effect on the BBB was shown. In vitro, endothelial cells HUVEC showed a change in their morphology and increased in number after exposure to static magnetic fields for 24 h a day; however, no effect was detected after shorter stimulation 82 . Although HUVEC cell line is not a BBB line, it is successfully used in BBB modeling 83 . An important marker of the BBB integrity-tight junction proteins expressionwas not evaluated, while endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) concentration-other sensitive markers in AD-were analyzed. eNOS expression increased after long-term exposure to the magnetic field, while NO level did not change 82 . In another study, to model the effect of tDCS on the BBB functions, a direct electrical current was applied to endothelial cells bEnd.3 84 . Anti-ZO-1 immunostaining of the cells demonstrated no damage to the cell monolayer integrity 84 . Probably, the intracellular parameters (e.g., NO, Ca 2+ ) are more sensitive than physiological parameters (e.g., transendothelial electrical resistance and BBB permeability to fluorescent tracers), however, the last ones are more significant as markers for the assessment of the NIBS effect.
We can conclude that the majority of the NIBS effects on BBB in the non-pathological model were toward BBB opening. In those studies when the BBB integrity was increased, pathological models were used [85][86][87] .

Discussion
There are many blind spots in AD pathology, which result in difficulties in the formulation of a desirable therapeutic effect of NIBS on the BBB. To answer this question, a translational approach is needed. However, translational studies of NIBS effects on the BBB in AD are challenging because, on the one hand, BBB studies are difficult in humans, and on the other hand, NIBS studies are difficult in in vitro and in vivo models. Lack of understanding of non-neuronal NIBS mechanisms in AD leaves multiple questions unanswered. Should one aim at BBB opening or closing in AD patients depending on the AD stage? Is the BBB state associated with the changes in cognitive performance? Is NIBS-induced BBB opening similar to the mechanism of the BBB disruption in neurodegeneration? Is NIBS effect in AD similar to NIBS effect in other conditions where BBB is compromised, such as multiple sclerosis 11,88,89 , Diabetes Mellitus 90 , etc. We believe that more studies should address the question of NIBS effects on the BBB as in AD, as well as in healthy aging. We believe that the parameters of a model should be chosen with an emphasis on its sensitivity to reveal possible fine NIBS effects on the BBB. We summarize candidate parameters for the future BBB investigation in cells, animals, and humans, in Table 3. We argue that in order to extrapolate the results of in vitro and in vivo animal works to human participants, one has to study NIBS effects on key components of AD pathology such as extra-and intracellular ion concentration changes, transendothelial resistance, and the proteins, primarily, Aβ and tau. At the same time, NIBS effects on transendothelial resistance and other in vivo parameters should be probed not only in intact animals but also in AD animal models. Yet another point for the NIBS effects on the BBB in AD is that additional investigation may be needed to assess NIBSinduced BBB mitigation in advanced AD stages for safety reasons. Considering that BBB mitigation using transcranial ultrasound stimulation (TUS) is already performed in clinical studies 91,92 , we   believe that the approaches used for the BBB assessment in such studies may be extrapolated to TMS/TES studies.
Conclusion NIBS techniques are widely tested as an alternative and an addition to pharmacological therapy in AD 8,9,69 . In this paper, we draw attention to the probable non-neuronal effects of NIBS such as BBB changes, both its opening and closing, and highlight its importance in AD. On the one hand, the BBB is vulnerable in AD, and its further deterioration might aggravate the pathology. On the other hand, BBB opening may be beneficial in AD in some cases for the purpose of drug delivery. We illustrate our point using evidence from human, animal, and cellular models (Fig. 2). The adaptation of NIBS protocols to re-and de novo investigating its effects on the BBB may be an important direction of the research in the field. We suggest that NIBS effects on the BBB should be investigated more considering that a large pool of cerebral blood vessels is located on the surface of the cortex, and is well accessible for NIBS.
Received: 21 December 2022; Accepted: 16 March 2023; Fig. 2 Scheme of the non-invasive brain stimulation (NIBS) action on blood-brain barrier (BBB) in Alzheimer's disease (AD). To receive noninvasive brain stimulation representative data, translational approach can be used including human, animal, and cellular models.